Fucosyltransferase 2 inhibitors: Identification via docking and STD-NMR studies

Fucosyltransferase 2 (FUT2) catalyzes the biosynthesis of A, B, and H antigens and other important glycans, such as (Sialyl Lewisx) sLex, and (Sialyl Lewisy) sLey. The production of these glycans is increased in various cancers, hence to design and develop specific inhibitors of FUT2 is a therapeutic strategy. The current study was designed to identify the inhibitors for FUT2. In silico screening of 300 synthetic compounds was performed. Molecular docking studies highlighted the interactions of ligands with critical amino acid residues, present in the active site of FUT2. The epitope mapping in ligands was performed using the STD-NMR experiments to identify the interactions between ligands, and receptor protein. Finally, we have identified 5 lead compounds 4, 5, 26, 27, and 28 that can be studied for further development as cancer therapeutic agents.

Multifrequency STD NMR Unveils the Interactions of Antibiotics With Burkholderia multivorans Biofilm Exopolysaccharide

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aβo–PrPC binding and downstream pathways

A new approach combining virtual screening, 19F and STD NMR, and biochemical assays using hiPSC and targetting multiple pathways involving Aβ, PrPC and Tau provides a more effective strategy for Alzheimer's disease drug discovery than Aβ only approach.

Saturation Transfer Difference NMR Spectroscopy Using Glycopolymers

<p>We report the use of Saturation Transfer Difference (STD) NMR spectroscopy to observe the interaction of various phenylboronic acids (PBAs) with synthetic glycopolymers presenting galactose and glucose. After optimizing experimental parameters to maximize spin diffusion, the binding of boronic acids to the glycopolymers was examined using STD NMR. Efficient amplification factor build-up curves which were used to generate an epitope map for the boronic acid binding to the glycopolymers. STD-NMR was also used to detect the interaction between indole and a galactosylated glycopolymer, providing an indole-based view of this C-H – π interaction.</p>

Saturation Transfer Difference NMR Spectroscopy Using Glycopolymers

<p>We report the use of Saturation Transfer Difference (STD) NMR spectroscopy to observe the interaction of various phenylboronic acids (PBAs) with synthetic glycopolymers presenting galactose and glucose. After optimizing experimental parameters to maximize spin diffusion, the binding of boronic acids to the glycopolymers was examined using STD NMR. Efficient amplification factor build-up curves which were used to generate an epitope map for the boronic acid binding to the glycopolymers. STD-NMR was also used to detect the interaction between indole and a galactosylated glycopolymer, providing an indole-based view of this C-H – π interaction.</p>

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.

Spin Diffusion Transfer Difference (SDTD) NMR: An Advanced Method for the Characterisation of Water Structuration Within Particle Networks

<p>Saturation transfer difference (STD) NMR spectroscopy is a well‑known ligand‑based solution NMR technique used extensively for ligand epitope mapping, the identification of the nature of ligand binding sites, and the determination of ligand binding affinity. Recently, we have shown that STD NMR can be also applied to monitor changes in bound water during gelation of particulate dispersions. However, this technique is strongly dependent on gelator and solvent concentrations and does not report on the degree of organisation of the solvent within the particle network. This obscures the detailed understanding of the role of the solvent on gelation and precludes the comparison of solvation properties between dispersions prepared under different experimental conditions. In this work we report a novel STD NMR method to characterise the degree of solvent structuration in carbohydrate-based particulate dispersions by demonstrating for the first time that, for solvents interacting with large particles, the spin diffusion transfer build‑up curves can be modelled by the general one‑dimensional diffusion equation. Our novel approach, called Spin Diffusion Transfer Difference (SDTD) NMR, is independent of the gelator and solvent concentrations, allowing to monitor and compare the degree of solvent structuration in different gel networks. In addition, the simulation of SDTD build-up curves report on minimum distances (<i>r</i>) and spin diffusion rates (<i>D</i>) at the particle‑solvent interface. As a case study, we have characterised the degree of structuration of water and low molecular weight alcohols during the alcohol‑induced gelation of TEMPO-oxidised cellulose hydrogels by SDTD NMR, demonstrating the key role of water structuration on gel properties. SDTD NMR is a fast, robust and easy-to-implement solution NMR protocol that overcomes some of the limitations of the classical STD NMR approach when applied to the study of solvation. This technique can be readily extended to characterise the solvent(s) organisation in any type of particulate gels. Hence, the SDTD NMR method provides key insights on the role of water in the mechanism of gelation and the macroscopic properties of particulate gels, of fundamental importance for the design of soft matter materials with tuneable properties. </p>